This invention concerns an improved method for the determination of sialic acid in plasma or serum which is less time-consuming, less expensive, less variable from sample to sample and less dependent upon the skill and experience of the person performing the test. Much work has been done which indicates that elevated sialic acid content in blood sera of a patient is an indication of the presence of cancer. For example, U.S. Pat. No. 4,146,603 to Davidson, et al. discloses and claims a fairly complex series of procedures whereby elevated sialic acid content is a determinant with respect to cancer specific determinations.
MacBeth and Bekesi, Cancer Res. 22:1170-1176 (1962) measured plasma glycoproteins and found galactose and mannose values were seen in breast cancers without metastases. Kloppel, et al., Proc. Natl. Acad. Sc. 74:3011-3013 (1977) reported 2.5-fold increases of serum sialic acid glycolipids in mice bearing transplantable mammary carcinomas and 2-fold increases in human carcinoma patients. The method involved column chromatographic separation of the gangliosides. A minimum of 1 ml whole blood was required. Kloppel, et al., Am. J. Vet. Res. 39:1377-1380 (1978) also reported increases of sialic acid in 92% of 24 dogs; however, a number of false positives were observed in dogs with other disorders. In leukemic AKR/J mice, Lengle, J. Natl. Cancer Inst. 62:1565-1567 (1979) found increased lipid bound sialic acid in their plasma and thymic lymphocytes. Lipid bound sialic acid levels were found increased in plasma and erythrocytes of humans bearing melanomas. Portoukalian, et al., Biochem. Biophys. Res. Commun. 85:916-920 (1978). Chromatographic separation and purification on columns was followed by evaluation on chromatoplates. Silver, et al., Cancer 41:1497-1499 (1978); Cancer Res. 39:5036-5042 (1979) have reported elevated serum sialic acid values in melanoma patients that were significantly related to the tumor burden. However, 36% of patients with observable tumors showed no elevated serum sialic acid. Hogan-Ryan, et al., Br. J. Cancer 41:587-592 (1980) reporting on total bound serum sialic acid in patients with breast cancer found elevations that corresponded with tumor stage.
One specific method over which the present invention is an improvement is disclosed in the American Association for Cancer Research Annual Meeting PROCEEDINGS Vol 21, March 1980 as Abstract No. 728 by Katopodis, et al. Briefly, this method requires that a 100 .mu.l plasma sample (reduced to 50 .mu.l) be extracted with 6 ml of a chloroform/methanol mixture, (2 to 1, volume to volume ratio). The lipid extract is then partitioned with 0.2 of its volume of water. The aqueous phase is evaporated in dryness and the residue redissolved in water. The lipid bound sialic acid is then purified by trichloroacetic acid-phosphotungstic acid precipitation and, after the removal of the supernatant from the resultant precipitate, the precipitate is determined by the Svennerholm and Miettien method (Svennerholm, Quantitative Estimation of Sialic Acid . . . , Biochem. Biophys, Acta 24, pp. 604-611 (1957)).
Another specific method over which the present invention is an improvement is disclosed in Katopodis and Stock, U.S. Pat. No. 4,342,567, issued Aug. 3, 1982. This method is similar to the foregoing but requires only about 50 .mu.l of sample rather than the 100 .mu.l required by the prior art method. The drying step is eliminated and there is no use of trichloroacetic acid. Phosphotungstic acid is used alone.
These specific methods suffer from a number of disadvantages including the following: the need for a precisely defined 44.7.lambda. starting sample; lipid bound sialic acid is lost during the tube inversion step creating reduced final values; precipitation of the lipid bound sialic acid with phosphotungstic acid is not complete, which is a particular problem when working with samples in which the amount exceeds normal values by only small amounts (e.g., early in cancer development); the rapidity of the test is limited by the 5 minutes waiting time after phosphotungstic acid addition and the cost of the test is not as low as is desirable.
Other methods over which the present invention is an improvement are disclosed in U.S. Pat. No. 4,701,418, issued Oct. 20, 1987 (Katopodis) and U.S. Pat. No. 4,748,128, issued May 31, 1988 (Katopodis). These methods involved the determination of the amount of sialic acid present in the sample by diluting a sample of human whole blood or plasma and then adding a mixture of a chlorinated lower alkyl hydrocarbon, such as chloroform, and a lower alkyl alcohol, such as methanol, mixing the resulting solution, and centrifuging the solution to form a substantially clear upper layer. A protein-precipitating agent is then added to a predetermined volume of the upper layer to precipitate lipid bound sialic acid. The protein precipitating agent disclosed is phosphotungstic acid.
After separating the precipitate from the supernatant, the precipitate is suspended in water and the amount of lipid bound sialic acid is determined by adding a resorcinol reagent and a mixture of butyl acetate and n-butanol and then measuring the absorbance of the resulting solution at 580 nm.
The present invention provides an improved method for determining the amount of sialic acid present in a sample of plasma or serum which in some embodiments is qualitatively and quantitatively more sensitive, and in others at least as sensitive as the methods of the prior art. Moreover, the present invention is more economical, more time efficient, more easily automated, and requires less labor and chemical reagents than the methods of the prior art. The procedure of the present invention differs significantly from known methods in that the present invention has eliminated the necessity for the protein precipitating agent and has reversed the proportions of the lower alkyl alcohol and the chlorinated lower alkyl hydrocarbon in the mixture used for extracting the sialic acid. The prior art discloses a mixture wherein the chlorinated hydrocarbon, typically chloroform, is dominant to the alkyl alcohol, i.e. 2:1 volume ratio. In contrast, the present invention uses a mixture of lower alkyl alcohol and chlorinated lower alkyl hydrocarbon wherein the alcohol to chlorinated hydrocarbon ratio is in the range from about 70:30 to about 85:15. In certain embodiments, the present invention also eliminates the need to use a butyl acetate/n-butanol mixture, which is corrosive and difficult to handle. Table 1 sets forth results obtained by others using the methods of U.S. Pat. No. 4,342,567 and it illustrates the variability obtained when samples from normal subjects were tested.
TABLE I ______________________________________ RESULTS OBTAINED BY DIFFERENT LABORATORIES USING THE METHOD OF U.S. Pat. No. 4,342,567 NORMAL SAMPLES RANGE MEAN UPPER LIMIT ______________________________________ 15.0-20.0 17.5 20.0 (1) 12.8-16.8 14.8 16.8 (2) 11.6-19.7 15.7 19.7 (3) 11.6-19.1 15.4 19.1 (4) 15.0-25.0 20.0 25.0 (5) 11.1-15.7 13.4 15.7 (6) 16.4-26.6 21.5 26.6 (7) NO INFO 15.3 NO INFO (8) NO INFO NO INFO 17.2 (9) 12.6-17.2 14.9 17.2 (10) 11.9-26.2 19.1 26.2 (11) 15.5-22.5 19.0 22.5 (12) 8.7-18.5 13.6 18.5 (13) 10.9-18.9 14.9 18.9 (14) 10.0-21.0 15.5 21.0 (15) MEAN- 12.3-20.6 16.4 18.2 ______________________________________ (1) KATOPODIS AND STOCK, U.S. Pat. No. 4,342,567 (2) CHEN SHUPAN et al., J. SHANGHAI MED. VOL. 6, 1983 (3) A. M. DNISTRIAN et al., CLINICAL CHEM. 27(10) 1981 (4) S. KAKARI et al., ANTICANCER RES. 4, Suppl. 1:3-6, 1984 (5) L. SANTAMARIA et al., MED. BIOLOGIE ENVIR. VOL. 12, 1984 (6) A. M. DNISTRIAN et al., AACR VOL. 23, 609, 1982 (7) P. KOSMIDIS et al., ASCO, VOL. 2, C1, 1983 (8) D. MUNJAL et al., FED. PROC., VOL. 42(3), March 1983 (9) K. M. ERBIL et al., CL, CHEM. 29, VOL. 6(194), 1983 (10) CHEN SHUPAN et al., CHIN. J. OBSTET. & GHY., 18(4):235-38 1983 (11) L. SALVAGNO et al., 13 INTL. CONG. OF CHEMO., 1983 (VIENNA) (12) L. SALVAGNO et al., I. OF CANCER RESEARCH, 1983 (13) A. K. BHARGAVA et al., ASCO, VOL. 6, No. 2, 1984 (14) S. KAKARI et al., INTL. MEETINGS, SALONICA, GREECE, 1982 (15) T. WUSTROW, GERMAN CANCER CONGRESS, 25/6 GL 1983